Innate behaviors offer an excellent opportunity to explore how complex behaviors are organized in the nervous system and how they are programmed during development, because they are likely to result from the activation of developmentally specified neural circuits. Here, I propose a strategy to identify and functionally characterize the specific neuronal circuits involved in innate behaviors in fruit fly, drosophila. Especially, I will focus on the role of octopaminergic neurons, which are implicated in the modulation of various innate behaviors, by addressing the following questions: (1) What is the effect of acutely manipulating octopamine levels on aggressive and mating behaviors, and on general arousal? (2) Which octopamine receptors are involved in mediating the effects of octopamine on these behaviors? (3) Which octopamine-synthesizing neurons are important in these behaviors? (4) In which neurons are the relevant receptors required? To address these questions, I will use genetic tools to conditionally enhance or inhibit octopamine release from various subsets of octopaminergic neurons in living drosophila, and perform various behavioral assays. Thus, I will functionally dissect the role of octopaminergic neurons to understand how specific neural circuits control specific behaviors. In contrast to my previous research experience in neural development, the proposed research will focus on understanding of the function of neural circuits, which mediate innate behaviors. Therefore, this will be a good opportunity to extend my training from developmental, to functional and behavioral neuroscience.

Cellular identities are defined by distinct gene expression programs that are set early during development and maintained via epigenetic mechanisms based on e.g. histone and DNA methylation as well as chromatin associated Trithorax and Polycomb group proteins. In mammalian pre-implantation embryos and primordial germ cells (PGCs), extensive changes in DNA methylation and histone modifications take place during the time of reacquisition of pluripotency. Importantly, members of the Polycomb Repressive Complex 1 (PRC1), implicated in homeotic gene silencing, X inactivation and stem cell renewal, are dynamically expressed during these developmental stages. Although epigenetic reprogramming events are thought to play a key role in establishing pluripotency, the precise underlying molecular mechanisms remain elusive. I propose to address the role of PRC1 in epigenetic reprogramming. First, I will use mice conditionally deficient for PRC1 components to examine PRC1’s regulatory role for higher order chromatin formation and directing cell fate in PGCs and pre-implantation embryos. Second, I will identify PRC1 target genes by genome transcription profiling using mutant mice. Third, I will address the significance of the PRC1 associated histone H2A lysine 119 mono-ubiquitination activity for epigenetic gene silencing in mammals. Through these projects, I will greatly extend my scientific horizon from my previous work on the regulation of chromosome segregation in fission yeast to the molecular mechanisms of epigenetic reprogramming in multi-cellular organisms. The proposed research will give me a thorough training in many aspects of epigenetic research and mouse molecular genetics.

Chromosome condensation is an essential process that ensures proper segregation of the genome during mitosis and meiosis. In vertebrate cells, two different condensin complexes, known as condensin I and condensin II, play central roles in this process. It remains unknown exactly how the two complexes are regulated and how their functions are coordinated during the cell cycle. In this proposal, I will take biochemical and cell biological approaches to this problem by using Xenopus egg cell-free extracts and human tissue culture cells. First, I will determine the distribution and dynamics of condensins I and II from prophase through metaphase. Their relative distributions on the metaphase chromatid axis will be assessed at high resolution by deconvolution and electron microscopy. Second, I will investigate the mechanism by which the recruitment and activity of condensins I and II are regulated by mitotic kinases. The hypothesis will be tested that different cyclin-Cdk complexes sequentially phosphorylate the two condensin complexes and thereby orchestrate their actions. Third and finally, I will test the idea that condensins may have a critical role in the functional organization of the interphase nucleus, with a major focus on its potential involvement in checkpoint activation and DNA repair. During my thesis work, my primary approach was genetics in fission yeast. The current application proposes to take biochemical and cell biological approaches in vertebrate cells. I believe that these new experiences will be important not only to deepen my understanding of chromosome function but also to broaden my scientific and technical scopes.

The number of species on the earth is estimated to be tens of millions, and the evolution of genes/genomes which resulted in this diversity of organisms is one of the most interesting topics in biology. I plan to work on cichlid fishes, instead of medaka, which have evolved astonishing variation within a very short period of time, in order to find out what types of genomic/genetic changes have been evolved to achieve this diversity. Because colours and colour patterns on the body surface are among the most divergent and readily recognizable features of animals, I chose pigment-cell-associated genes as the best targets of this research proposal. The aim of this study is to identify the number and chromosomal locations of genes responsible for body-colour determination as well as mating preference in cichlids for later positional cloning and functional studies. Several pairs of fishes which 1) have distinct body colour differences, 2) reach maturity quickly, 3) lay enough eggs for fine locus/QTL mapping, and 4) are closely related to each other and whose F1s are known to be fertile (e.g., The Normal and Gold morphs of A. citrinellus) are crossed/hybridised in the laboratory, and the phenotypes (i.e., body colour and mating preference) of their backcross siblings are analysed to be grouped. I perform AFLP on their genomic DNAs and phenotype-specific bands are used for STS construction. Linkage and recombination distances between the responsible locus/QTL and STSs are evaluated. Tens of backcross siblings are enough for this rough mapping, but I will construct maps as fine as possible for future gene identification by the forward genetic approaches.

The refinement of synaptic connections is crucial for the development and function of the nervous system. It is coordinated by a combination of synapse formation and disassembly of previously formed connections, and abundant evidence supports that both of these processes require neural activity. Our preliminary experiments have shown that myocyte enhancer factor 2 (MEF2), an activity-regulated transcription factor, acts as a negative regulator of synapse number in developing cultured hippocampal neurons. This finding suggests two possible effects of MEF2 on synapse development: 1) MEF2 decreases synaptogenesis or 2) MEF2 promotes active synapse disassembly. To distinguish between these possibilities, we plan to utilize a regulated gene activation system that will allow temporal control of MEF2 activation at the time of synapse development. In addition, to characterize the molecular mechanisms by which MEF2 mediates synapse development, we will employ a genome-wide screen to identify neuronal targets of MEF2. Finally, we propose to generate transgenic mice to explore the physiological role of MEF2 in synapse development and subsequent effects on animal behavior. My previous research has focused on identifying the molecular mechanisms by which immune cells recognize changes in their environment. In the proposed research, I plan to analyze the signal transduction pathways that operate in cells of the nervous system and mediate their responses to environmental stimuli. This research experience will provide me with a great opportunity, not only to acquire interdisciplinary knowledge and techniques, but also to investigate the fundamental principles that govern cell function.

Defining the DNA damage response at chromosome ends This project is aimed at understanding how cells detect and process dysfunctional telomeres. When telomeres are shortened in primary human cells, the DNA damage response is activated leading to apoptosis or senescence. Although this pathway is thought to be important for aging of human cells and contributes to certain aspects of human cancer, little information is available on how dysfunctional telomeres are detected. In this project, the molecular details of this response will be defined. The approach will use a combination of genetics, cell biology, biochemistry, and proteomics to define the proteins associated with dysfunctional telomeres and the signaling pathways involved. The research will provide me with training in cell biology and genetics and educate me in the areas of telomere biology, aging, and cancer.

Courtship behaviors are highly specialized, innate behaviors essential for propagation in higher animals. Pheromone recognition is the most important sense for this process in many animals, including many insects. Recently, a candidate pheromone receptor with a specific function in male courtship was identified in Drosophila, providing a precedent for a single pheromone receptor gene with a specific behavioral function. Moreover, the gene is closely related to seven additional genes, and it is likely that some or all of them function as additional pheromone receptors. I plan to characterize expression of four members of this pheromone receptor gene family, and I shall determine their specific roles in courtship behavior. At present, little is known about the relationship of individual pheromone receptors and mating behaviors they control. Thus, my studies will establish a novel catalogue of innate behaviors, which are controlled by specific pheromone receptors. Moreover, these investigations will shed light onto the neurobiological and molecular processes of complex behaviors, and they will generate new tools for future studies on their neural circuitry. I have previously studied chloroplast RNA editing, a completely different topic than the one proposed here. However, I have recently become very interested in the general area of neurobiology, with a specific interest in processes that control innate or learned behaviors. Even though this new field is quite different from my previous work as a graduate student, my expertise in molecular techniques is readily applicable to the characterization of pheromone driven processes.

The phylogenetic position of Xenoturbella has long remained obscure, and although they were recently reported as deuterostomes, further studies are essential to confirm their phylogenetic position. By revealing their life cycle and gathering the traits showing the evolutionary history of the animal, I aim to elucidate the phylogenetic position of the animal. If they are confirmed to be deuterostomes, new knowledge of deuterostome evolution will be obtained through this project. The early history of deuterostomes, to which we belong, remains unclear, but Xenoturbella may represent the simple body plan of the last common ancestor of the deuterostomes. Research on their morphology, gene expression patterns, and embryology should reveal important features of the early evolution of deuterostomes and the body plan of their common ancestor. The first important task will be examining the reproductive cycle of Xenoturbella. The breeding season will be determined followed by induction of spawning and/or embryo release. Developmental stages will be reared to adults. Examples of each stage will be fixed and analyzed by several morphological methods. In addition, expression patterns of developmental regulatory genes will be investigated to better understand the structure of Xenoturbella. Molecular studies will be a new experience to me. As molecular techniques become essential to most areas of biology, it is vital for me to acquire them early in my career. I will also learn ecological methods and establish a long-term culture system for Xenoturbella. Long-term culture of live specimens will enable observation of natural spawning, mating behavior, fission, and regeneration.

The development of multicellular organisms occurs in four dimensions, the three axes of space and a fourth axis of time. While a great deal is known about the fundamental mechanisms of spatial pattern formation, much less is known about temporal patterning during development. The objective of my project is to unravel the molecular mechanisms underlying developmental timing. To achieve the goal, I plan to reveal the molecular circuits functioning with the hunchback-like-1 (hbl-1) transcription factor in C. elegans. hbl-1 has unique features because it is a target of temporal microRNAs and its orthologues are essential for regulation of developmental timing in C. elegans and Drosophila. I propose to: (1) screen for mutations that suppress hbl-1 and clone the modifying genes, (2) perform a DNA microarray analysis to identify downstream targets of hbl-1, and (3) functionally characterize these genes at molecular level. Since hbl-1 is evolutionally conserved in structure and function, this project will provide a solid basis for understanding evolutionally conserved mechanisms of developmental timing. The research field (developmental timing, transcription factor, microRNA), the methodology (genetic modifier screening, genome-wide analysis of gene expression) and the model organism (C. elegans) are all different from my previous work (cell biology of actin cytoskeleton and insect molecular endocrinology). I have not experienced these techniques, and they are important for understanding complex developmental events in this post-genomic era. This experience will be very useful in the future when I establish my own research program and educate students as an independent investigator.

My past studies mainly focused upon chemical reactions catalyzed by proteins in vitro. In order to analyze the reaction mechanisms of some enzymes, I have been trained in X-ray crystallography, the synthesis/assays of chemical compounds, and protein expression/purification. I have succeeded in analyzing the reaction mechanisms of macrophomate synthase (natural Diels-Alderase) and ACC deaminase by mutagenesis from structural information combined with chemical methods. Expertise in enzyme chemistry will make important contributions to studies of cellular function. I am going to focus on the biochemical and structural studies of protein-protein interactions and multi-component assemblies of scaffold proteins in a signal-transduction pathway in the host laboratory. Scaffold proteins play a central role in integrating the input and output of signaling systems and are increasingly the focus of cancer research. I think that my expertise in chemistry, crystallography and NMR spectroscopy is a good starting point to explain protein-protein interactions. One of the most efficient methods to identify and evaluate protein-protein interactions is using NMR spectroscopy prior to X-ray analysis. In parallel with crystallographic studies I will carry out HSQC measurements NMR spectroscopy will also be a useful tool in judging whether the recombinant proteins are folded correctly and suitable for crystallization. Biological techniques that will be available in the host laboratory range from tissue culture (and cell based assay employing confocal microscopy) to in silico drug design.

Metastasis accounts for a large part of cancer malignancy. Invasion is the initial step of metastasis, and acquiring aggressive invasiveness is important for the subsequent dissemination of tumor cells into circulation. Changes in the invasiveness of tumor would require harmonized changes in the action of several factors either inside, surface or outside of the tumor cell, indicating that exhaustive analyses like gene expression profiling are effective in studying this process. Recently, it has been shown that a transcription factor Twist, which plays major roles in embryonic morphogenesis, also plays an essential role in the regulation of tumor invasion. Twist enhances tumor invasiveness possibly by inducing an epithelial-mesenchymal transition (EMT) of tumor cells. In the proposed study, I will set up a system to express Twist in an inducible manner in cells of the transplanted tumor. It is expected that Twist triggers an EMT of the tumor cells and promotes invasion of the transplanted tumor. It will therefore provide an experimental system to study how the invasiveness of tumor is enhanced. Subsequently, to address the molecular basis supporting the enhancement of invasiveness, I will analyze the time-scale changes in the gene expression profile of the tumor cells after Twist expression. As a PhD student, I have studied p53-mediated apoptosis, an intracellular mechanism concerned with tumorigenesis. In contrast, through this fellowship experience, I will mainly study the behavior of tumors in the tissue context. Therefore, this experience will particularly help me in obtaining a comprehensive view on the process of malignant tumor progression.

MicroRNAs (miRNAs) are a family of small non-coding RNAs that negatively regulate gene expression in a sequence-specific manner. They regulate aspects of development in multicellular organisms including Arabidopsis. However, our knowledge about roles of miRNAs in plant morphogenesis is still rudimentary. I will elucidate the biological role of the post-transcriptional regulation of the TIR1 gene and its homologs by an Arabidopsis miRNA, miR393 with respect to auxin signaling pathways. Auxin is an essential phytohormone that influences aspects of plant morphogenesis. The TIR1 gene is a pivotal positive regulator in auxin signaling pathway. Although the biological functions of the TIR1 homologs have not yet been characterized, similarities of their amino acid sequences suggest that the homologs are also required for auxin signaling. Therefore, miR393 is expected to negatively regulate a wide rage of auxin responses by eliminating its targeted transcripts. This research proposal will contribute to defining an important regulatory system in the auxin response pathway. As described above, I am interested in the mechanism of cell division control in plant development. Auxin is the most important phytohormone to control cell proliferation. An understanding the mechanism of auxin signaling is a prerequisite to unraveling the molecular mechanism of cell division control. In addition, the post-doctorial training will allow me to acquire technical skills and scientific knowledge analyzing multicellular system of plants, although my doctoral training has been primarily in plant cell biology.

Stereo vision is a model system for investigating the neural mechanism of information processing, because the computational task of the stereoscopic system is well-defined: a single neural representation of the 3D scene is derived from a pair of slightly disparate 2D representations for the left and right retinal images. The neural mechanism of the initial level of stereo processing is fairly well understood with a simple model, but it does not account for all aspects of stereoscopic depth perception. The neural representation must be further processed at higher extrastriate visual areas in order to fully account for the perceived stereoscopic depth. However, the neural mechanism of processing the initial representation of binocular disparity into a higher representation of stereoscopic depth is yet an open question. This project is aimed to bridge this gap. Computational modeling of a neuronal network of V1 and an extrastriate area will provide testable predictions of neural responses to grating patterns of various combinations of spatial frequencies and disparities, as well as their compounds. Then we will test these predictions by single-cell recordings of the responses of visual cortical neurons in awake, behaving monkeys. The research plan for this application is novel to the applicant in that it merges the theoretical and experimental approaches that he has so far experienced. Such comprehensive study of visual information processing is essential for a better understanding of the neural mechanism of complex cognitive functions of organisms such as perception and behavior. This is the direction of research that the applicant aspires to for a professional career.

Establishment and maintenance of cell polarity belong to the most basic problems in cell and developmental biology. Polarity of cells is a necessary precondition for the asymmetric distribution of intracellular components. In plants, the polar localization of PIN proteins, which are recently reported by Dr Jiri Friml and colleagues, provides an excellent marker of cellular polarity. So far, the mechanism, which regulates cell polarity in plants is only poorly understood. In the presented project I propose a forward and reverse genetic approach in the model system Arabidopsis to specifically identify the factors regulating cell polarity in plants. The idea for the project on cell polarity emerged from my previous work, when two proteins (ACR4-GFP and ALE2-GFP), which I identified, represent themselves as novel markers for cellular polarity. For this project, an excellent marker for cell polarity as well as techniques in cell biology is essential. Dr. Friml’s group fulfils both of these requirements. Using well established genetic and cell biology methods in host laboratory together with an unique set of cell polarity markers (PIN-GFP, anti-PIN antibodies as well as ACR4-GFP and ALE2-GFP markers), components essential for generation and maintenance of cell polarity in plant will be identified. Through this project, I aim to acquire an expertise in cell biology. Beside, ZMBP Tübingen is a progressive institution with an unique organization structure. This will greatly add to my learning how to organize independent research. Thus training through this fellowship will give me an excellent opportunity for my professional growth and greatly help to achieve my scientific goal.

The goal of my proposed project is to understand the role of molecular chaperones in the maintenance of the prion state. I will use Saccharomyces cerevisiae as a model system and focus on the yeast prion [RNQ+] maintenance modulated by Hsp70 Ssa, using various analysis methods of genetics, cell biology, molecular biology, biochemistry, and physical chemistry. Firstly, I will isolate SSA mutants defective in maintenance of the prion state in yeast, and then characterize these effects using cell biological techniques. Next, to understand the mechanism at a molecular level, I will purify these mutants and characterize them in vitro assay systems using biochemical techniques. This project will provide us both fundamental understanding of the prion propagation mechanism in a cell and thus potentially have important implications for medicine and biotechnology. I have studied physical properties of protein in vitro using physicochemical and physical techniques, whereas in my proposed project, I will study protein functions in the cell using cell biological methods. Thus, there are significant differences between my previous research and my proposed project in both research direction and method. I am eager to acquire expertise in the cell biological and genetics field, in addition to the physicochemical and biophysical skills I already have. I think that a cell biological and molecular biological approach combined with in vitro biochemical and physicochemical techniques is important for unraveling the complex functions of living organisms. The HFSP fellowship will provide me a great chance to obtain training in a new area of research and to perform combined analysis approaches.

1) Proposed project: This project is the first approach to genetically dissect the mechanisms of metastasis in Drosophila. To identify the genes regulating metastasis, I will perform a large-scale genetic screen using a Drosophila cancer model. I will utilize a Drosophila model of benign tumor established in Dr. Tian Xu’s laboratory, and conduct a genetic screen to identify mutants acquiring metastatic features in these tumors. I will genetically isolate responsible genes for the ‘metastasis promoting’ mutations, and dissect the signaling pathways of metastasis both genetically and molecularly. I believe that it would bring a breakthrough in this research field. 2) How it differs from my previous research experience: So far I have studied the mechanisms of cell death in mammals and Drosophila. Taking advantage of powerful genetics of Drosophila, I propose a novel approach dissecting the mechanisms of metastasis through genetics. Metastasis is achieved by several stepwise progressions of tumor cells including cell growth, invasion and cell migration. This line of biological phenomenon can be accomplished independently of cell death machinery, indicating this research field is completely different from all my previous experiences. 3) How it will serve my professional growth: Through this project, I will experience highly complicated genetics and strategies against complex biological phenomena in whole animals. In addition, the genetic approach to understand the mechanisms of metastasis will provide me an experience to study various biological features of cells and their signaling pathways that I have never studied. These aspects will strongly support my professional growth.

1) To become functionally active, newly synthesized protein chains must fold to unique three-dimensional structure, but regulated unfolding also is critical during the life cycle of many proteins. Some unfolding processes show surprising similarities. For instance, many proteins in degradation and membrane translocation are targeted for unfolding by terminal signal sequences and translational movement of the substrate protein often occurs with unfolding. Taking together the previous results, the susceptibility of proteins to unfolding by protease and translocase depends on the proteins' stability against global spontaneous unfolding and, more importantly, on their local structure adjacent to the targeting signal. I assume that the unfoldases trap local unfolding fluctuation in the part of the substrate structure adjacent to the targeting signal effectively pulling at the polypeptide chain. To test this hypothesis, I will measure both the local unfolding fluctuations adjacent to the targeting signal in substrate proteins by using NMR spectroscopy as well as the physical stability of substrate proteins against pulling in atomic force microscopy experiments. 2) & 3) I have worked on the biophysical side of molecular chaperone which assists protein folding and my host laboratory has worked on biochemical and molecular biological as well as biophysical aspects of in vivo protein unfolding. Although protein unfolding is an opposite phenomenon to protein folding, my biophysical background will allow me to make unique contribution to biologically important in vivo protein unfolding and I will broaden my research experience to include in vivo protein unfolding.

In the proposed project, I will investigate fundamental questions concerning the link between behavior and neuronal activity in the brain. In order to address these questions, I will develop novel techniques for combining of electrical and optical recording from rodent cerebellum in vivo. The activity of the cerebellar cortical network will be visualized using voltage-sensitive dyes and a high-speed cooled CCD camera. At the same time, cellular and synaptic activity will be investigated using patch-clamp recordings from single neurons combined with two-photon microscopy. Changes in the activity of cerebellar cortex induced by sensory stimulation will be captured by these techniques. I will also try to induce long-term depression at the parallel fiber and Purkinje cell synapse by applying a paradigm of classical conditioning in order to test whether simple motor learning is associated with plastic changes at the cerebellar synapses. Until now I have been working on synaptic transmission in the brain slice preparation using patch-clamp methods. The framework of the proposed project is consistent with that of my previous work in the sense that it investigates synaptic modulation using electrophysiology. However, the project also offers me a transition from the slice preparation to the intact brain in vivo, with imaging techniques paired with electrophysiology. These technical developments will allow me to relate the synaptic dynamics that I have been studying to the function of neural networks and ultimately to behavioral events. This project will therefore enable me to expand the scope of my research and gain a wider range of experience in the field of neuroscience.

Sister chromatid cohesion is the process whereby the two chromatids produced by DNA replication are held together until the mitotic cell division event. This process is mediated by the multi-subunit protein complex called cohesin. Recent biochemical analyses suggest that cohesin's subunits form a ring structure. The ring hypothesis proposes that sister DNA molecules are embraced within the cohesin ring. According to the hypothesis, the cohesin ring must open to allow chromosomal DNA to enter the ring. After cohesion has been established during S-phase, the ring needs to be 'locked' until anaphase to hold sister chromatids together. The 'locked' ring can only be opened by proteolytic cleavage of cohesin's Scc1 subunit. In comparison to cohesion disruption, little is known about how the cohesin subunits assemble during S-phase when cohesion is established. Furthermore, there have been no studies addressing the loading of cohesin onto DNA or chromosomes in vitro. My research activity will be to characterize the cohesion establishment process in vivo and in vitro. First, assembly or the exchange of the cohesin subunits will be studied in vivo. In parallel, purified cohesin subunits will be prepared to determine the molecular basis of assembly coupled with crystal structure determination. Consequently, in vitro loading of cohesins onto DNA and establishment will be examined. These studies are different from the previous ones that I am aiming to study cohesion at molecular and cellular level. Nonetheless, my training of structural biology will help me greatly to carry out this project. Thus, HFSP award should provide me the firm foundations for my professional goal.

1) My proposed project focuses on one of the GTPases of the Ras superfamily that are involved in membrane traffic. In particular on the human Arf-like GTPase, Arl8, a member of the Arf family whose other members recruit vesicle coats and other effectors to the endoplasmic reticulum and Golgi apparatus. However, Arl8 is unexpectedly located to lysosomes and late endosomes. The existence of such an Arf family GTPase in the endocytic pathway was not anticipated, but its importance is suggested by it being conserved from plants to humans. This project is intended to examine the function of Arl8, and to identify the effectors that it recruits to membranes. 2) My previous work was in the elucidation of molecular mechanisms of morphogenesis in plants. For my postdoctoral training I will change my research field to yeast and mammalian cells. Before, I focused on transcription factors and identifying their target genes, I will change my focus onto the membrane traffic system using biochemical and cell biological approaches. 3) I will move my research field from the multicellular plant to the unicellular yeast and cultured human cells. This shift will give me a new and different point of view to see eukaryotic organisms. The cell biological techniques and the methods of analyzing protein-protein interactions used in the proposed institute are now indispensable for understanding biological phenomena, and the scientific activity in the host laboratory is very high. Acquiring these techniques and working in such a favorable circumstance will develop my ability for my professional career and give me a great opportunity to discuss with people at a high level.